UCL Eastman Dental Institute

Real Time Quantitative PCR And Allele Discrimination

Introduction

Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. In contrast, an endpoint assay (also called a “plate read assay”) measures the amount of accumulated PCR product at the end of the PCR cycle. Applied Biosystems has developed two types of chemistries used to detect PCR products using Sequence Detection Systems (SDS) instruments, the TaqMan® chemistry (also known as “fluorogenic 5´ nuclease chemistry”) and SYBR® Green I dye chemistry.

TaqMan® Chemistry

The TaqMan chemistry uses a fluorogenic probe to enable the detection ofa specific PCR product as it accumulates during PCR cycles.

Assay Types That Use TaqMan Chemistry

The TaqMan chemistry can be used for the following assay types:Quantitation, including:

One-step RT-PCR for RNA quantitation

Two-step RT-PCR for RNA quantitation

DNA/cDNA quantitation

Allelic Discrimination

Plus/Minus using an IPC

SYBR Green I Dye Chemistry

The most important difference between the TaqMan and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry will detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore essential for accurate results.

Assay Types That Use SYBR Green I Dye Chemistry

The SYBR Green I dye chemistry can be used for the following assay types:

One-step RT-PCR for RNA quantitation

Two-step RT-PCR for RNA quantitation

DNA/cDNA quantitation

How TaqMan Sequence Detection Chemistry Works

The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR. Here’s how it works:

Step Process

An oligonucleotide probe is constructed containing a reporter fluorescent dye on the 5´ end and a quencher dye on the 3´ end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET) through space.

If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5´ nuclease activity of Taq DNA polymerase as this primer is extended.

This cleavage of the probe:• Separates the reporter dye from the quencher dye, increasing the reporter dye signal.• Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand.Thus, inclusion of the probe does not inhibit the overall PCR process.

Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.

What Is A Quantitation Assay?

A Quantitation Assay is a real-time PCR assay. It measures (quantitates) the amount of a nucleic acid target during each amplification cycle of the PCR. The target may be DNA, cDNA, or RNA. There are three types of Quantitation Assays discussed in this chemistry guide:

Absolute Vs. Relative Quantitation

When calculating the results of your quantitation assays, you can use either absolute or relative quantitation.

What Is Absolute Quantitation?

The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve.

What Is Relative Quantitation?

A relative quantitation assay is used to analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated controlsample).

What Is An Allelic Discrimination (AD) Assay

An allelic discrimination (AD) assay is a multiplexed (more than one primer/probe pair per reaction), end-point (data is collected at the end of the PCR process) assay that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleic polymorphism (SNP) site in a target template sequence. The actual quantity of target sequence is not determined.

For each sample in an AD assay, a unique pair of fluorescent dye detectors is used, for example, two TaqMan® MGB probes that target an SNP site. One fluorescent dye detector is a perfect match to the wild type (allele 1) and the other fluorescent dye detector is a perfect match to the mutation (allele 2).